The present invention relates to a microchip-based electrospray ionization device and column with affinity adsorbents and a method of using the device and column.
Although efforts to evaluate gene activity and to explain biological processes including those of disease processes and drug effects have traditionally focused on genomics in the past two decades, more attention has been paid to proteomics in recent years due to its offering a more direct, complete and promising understanding of the biological functions of a cell. Proteomics research is targeted towards a comprehensive characterization of the total protein complement encoded by a particular genome and its changes under the influence of biological perturbation. Proteomics also involves the study of non-genome encoded events such as the post-translation modification of proteins, interactions between proteins, and the location of proteins within the cell. The study of the gene expression at the protein level is important because many of the most important cellular activities are directly regulated by the protein status of the cell rather than the status of gene activity. Also, the protein content of a cell is highly relevant to drug discovery and drug development efforts since most drugs are designed to target proteins. Therefore, the information gained from proteomics is expected to greatly boost the number of drug targets. Current technologies for the analysis of proteomics are based on a variety of protein separation techniques followed by identification of the separated proteins. Currently, the most popular method for proteomics investigation is the use of high-resolution two-dimensional gel electrophoresis (2D-gel) to map the biological complexity at the molecular level, followed by in-gel proteolytic digestion and sensitive mass spectral techniques to identify the spots of interest. Complex biological materials typically contain hundreds of biological molecules plus organic and inorganic salts which preclude direct mass spectral analysis. Therefore, significant sample preparation and purification steps are required prior to proteolytic digestion and mass spectral analysis. Although 2-D gel is one of the most powerful methods in the current study of proteomics, this method suffers from the labor-intensive, time consuming, attendant analyte loss and limitation of staining sensitivity to detect the low abundance proteins or peptides. The 2-D gel method suffers from poor reproducibility. In addition, electrophoretic techniques are also plagued by a bias towards proteins of high abundance and the variation of solubility among the complex proteins.
Obviously, there is a need for direct and facile mass spectrometric detection for both major and minor proteins in heterogeneous samples. The significant demands evolving from both the rapid increase of new drug targets and the availability of vast libraries of chemical compounds also apply to the new technologies that can facilitate the screening process.
To avoid the aforementioned disadvantages of the 2-D gel technique, some microchip-based separation devices (arrays) have been developed for rapid analysis of large numbers of samples. Compared to conventional separation columns or devices, microchip-based separation devices (arrays) have higher sample throughput, reduced sample and reagent consumption, and reduced chemical waste. Such devices are capable of fast analyses and provide improved precision and reliability compared to the conventional analytical instruments. The liquid flow rates for microchip-based separation devices range from approximately 1 to 500 nanoliters (nL) per minute for most applications. Capillary electrophoresis (CE) and capillary electrochromatography (CEC) are the two major separation modes used for microchip-based devices. However, liquid chromatography (LC) is not a major separation mode for microchip-based devices and currently is limited to an infusion mode in some limited applications.
Recently, a chip-based proteomics approach has been introduced using biomolecular interaction analysis-mass spectrometry (BIA-MS) in rapidly detecting and characterizing proteins present in complex biological samples at the low- to sub-fmole level (Nelson et al., 2000 Electrophoresis 21: 1155-63). One of the most powerful techniques is surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technology which was commercially embodied in Ciphergens""s ProteinChip Array System (Merchant et al., 2000 Electrophoresis 21: 1164-77). The system (aluminum chip) uses chemically (cationic, anionic, hydrophobic, metal, etc.) or biochemically (antibody, DNA, enzyme, receptor, etc.) treated surfaces for specific interaction with proteins of interest, followed by selected washes for SELDI-TOF-MS detection. The power of the system incorporates straightforward sample preparation with on-chip capture (binding) and detection for protein discovery, protein purification, protein identification from small samples, allowing rapid analysis and assay development on a single platform. Compared to the classic methods of sample purification, the advantages of the Protein Chip system include:
1. on-line xe2x80x9cone-stepxe2x80x9d separation of a small amount of crude biological sources for high throughput analysis;
2. In situ clean-up which diminishes sample loss by eliminating non-specific binding, reducing analyte signal suppression;
3. Pre-concentration of the target molecules, increasing the detection sensitivity particularly for the minor targets compounds.
However, the SELDI-TOF-MS based Protein Chip system suffers from the inability to provide the primary sequencing and structure information for bio-polymers such as proteins and peptides, and for small compounds. It has limitations with respect to the quantitative analysis of analytes. It also has a limited detection level for analytes and limited range of proteins, since only a low number density of analyte is available at any small point on a array spot where the laser beam can hit and generate ions for detection. The detection levels will significantly decline for proteins with a molecular mass above 15-20 Kda.
Electrospray ionization (ESI) provides for the atmospheric pressure ionization of a liquid sample. The electrospray process creates highly-charged droplets that, under evaporation, create ions representative of the species contained in the solution. An ion-sampling orifice of a mass spectrometer may be used to sample these gas phase ions for mass analysis. Electrospray in front of an ion-sampling orifice of an API mass spectrometer produces a quantitative response from the mass spectrometer detector due to the analyte molecules present in the liquid flowing from the capillary. One advantage of electrospray is that the response for an analyte measured by the mass spectrometer detector is dependent on the concentration of the analyte in the fluid and independent of the fluid flow rate. The response of an analyte in solution at a given concentration would be comparable using electrospray combined with mass spectrometry at a flow rate of 100 xcexcL/min compared to a flow rate of 100 nL/min. D.C. Gale et al., Rapid Commun. Mass Spectrom. 7:1017 (1993) demonstrate that higher electrospray sensitivity is achieved at lower flow rates due to increased analyte ionization efficiency. Thus by performing electrospray on a fluid at flow rates in the nanoliter per minute range provides the best sensitivity for an analyte contained within the fluid when combined with mass spectrometry.
The increasing demand for more efficient and rapid separation techniques in many areas, especially for the pharmaceutical industry, has initiated research towards column consolidation and miniaturization. In recent years, such column consolidation has been achieved when porous polymer continuous beds or monoliths were introduced or invented. Hjertxc3xa9n, J. Chromatography, 473 (1989), 273-275 discloses a polymer gel continuous bed prepared by in situ polymerization of an aqueous solution of acrylamide derivatives. Svec and Frxc3xa9chet disclosed in 1994 and 1995 (U.S. Pat. Nos. 5,334,310 and 5,453,185) a continuous liquid chromatographic column containing a separation medium in the form of a macroporous polymer plug. Column miniaturization has also been achieved when a porous polymer monolith was prepared by radical polymerization in situ in a fused silica capillary. The development of fritless columns with a polymer-based porous monolith rather than conventional spherical beads has become more and more important since it meets the requirement of today""s micro-scale liquid chromatography and capillary electrochromatography as described by Peters et al., Analytical Chemistry, 70 (1998), 2288-2295; and Gusev et al., J. Chromatography A, 855 (1999), 273-290. It would be desirable to provide a microchip device integrated with the miniaturized and consolidated micro-columns/packings for proteomics research.
In an effort to overcome the above drawbacks to the prior art, the present invention provides a microchip-based ESI device including a miniaturized and consolidated micro-column and micro-column array having affinity chromatographic adsorbents, which offers higher selectivity and sensitivity, and more accurate qualitative analysis than prior disclosed protein chips and provides quantitative analysis of analytes. The ESI device also offers the capability of providing additional structure and primary sequence information for analytes. In an alternative platform, the microchip device has built-in or attached micro-columns containing an adsorbent in the form of a porous polymer monolith or a coated support. Both platforms and their combinations are used for the detection of complex protein samples and screening of combinatory chemical compounds. In addition, the platforms have potential uses for non-covalent binding in identifying protein-protein, protein-ligand interactions.
An aspect of the present invention is to develop a microchip-based on-line device for both the affinity capture of biomolecules and the electrospray ionization in coupling with a mass spectrometer.
Another aspect of the invention is to provide a method for using such device for affinity capture of biomolecules to meet the needs for the modern life sciences such as proteomics, drug discovery, clinical diagnostics and forensic science.
A further aspect of the present invention relates to an electrospray device having flow-contacting portions including an affinity chromatographic adsorbent.
A further aspect of the present invention relates to a method for analysis including: providing the electrospray device; and selectively immobilizing affinity ligands on the flow-contacting surface of the device.
A further aspect of the present invention relates to a method for analysis including: providing the electrospray device; selectively binding an analyte on the affinity chromatographic adsorbent by affinity capture; optionally, performing chemical, enzymatic, or physical treatment of the immobilized analyte; selectively desorbing the analyte; electrospraying the desorbed analyte; and passing the electrosprayed analyte to a detector.
A further aspect of the present invention relates to a chromatography column including an affinity chromatographic adsorbent.
According to the invention, these objects have been achieved by a microchip-based electrospray ionization device having an affinity chromatographic adsorbent. The device is the combination of a monolithic silicon microchip having a reservoir/nozzle array and a capillary tube/column in communication with one of the chip reservoir. The affinity adsorbent is immobilized in either the chip reservoirs/channels or the capillary tube, or in both. The affinity adsorbent is in the form of either a porous polymer monolith or a surface coating with affinity functions on the flow-contacting surfaces. The affinity chromatography of the present invention includes immobilized metal affinity chromatography (IMAC). The device and its use can achieve the simultaneous affinity capture of biomolecules and subsequent electrospray ionization for mass spectrometry analysis.